1. Cells were passaged every 3-4 days using the Gentle Cell Dissociation Reagent (100-0485, STEMCELL Technologies) to dissociate iPSC colonies into small cell clumps. Organoids derived from spontaneous aggregation (SA) and single-cell aggregation in a 96-well ultra-low attached plate (96W);UCOs culture Human iPSCs (IMR90-4, WiCell) were cultured on matrigel-coated culture dishes or 6-well plates with mTeSR1 plus media
2. mg/mL) for 30 minutes in a 37? incubator. Suspended iPSC clumps were resuspended in mTeSR1 plus media with 10 ?M of Y-27632 and gently pipetted using a 5 mL serological pipette. Small pieces of iPSC clumps were spread into the 1000 ?m of microwell (H2951000, MICROFIT);UCO generation was based on the SA organoid method with modifications
3. EBs were collected and cultured in 100 mm ultra-low attachment (ULA) dishes (4615, Corning) with mTeSR1 plus media for an additional day;Subsequently;Neural induction was started by replacing the neural induction media
4. On day 4, the EBs were transported to 6-well ULA plates, and 2.5 ?M IWP-2 (HY13912, MedChem Express) was added to the neural induction media. From days 10-14, the media were changed daily to neural progenitor cell (NPC) expansion media (Neurobasal A medium [10888-022, Gibco] containing B27 supplement minus VitA;Sigma and 10 ?M SB431542; S4317, Sigma) for 3 days